Parasites of the phylum Apicomplexa include many important human and veterinary pathogens such as Plasmodium (malaria), Toxoplasma (a leading opportunistic infection associated with AIDS and congenital neurological birth defects), and Eimeria (an economically significant disease of poultry and cattle). Recent studies have identified an unusual organelle in these parasites: a plastid that appears to have been acquired by secondary endosymbiosis of a green alga. Here we show that replication of the apicomplexan plastid (apicoplast) genome in Toxoplasma gondii tachyzoites can be specifically inhibited using ciprofloxacin, and that this inhibition blocks parasite replication. Moreover, parasite death occurs with peculiar kinetics that are identical to those observed after exposure to clindamycin and macrolide antibiotics, which have been proposed to target protein synthesis in the apicoplast. Conversely, clindamycin (and functionally related compounds) immediately inhibits plastid replication upon drug application-the earliest effect so far described for these antibiotics. Our results directly link apicoplast function with parasite survival, validating this intriguing organelle as an effective target for parasiticidal drug design.

In order to characterize the delayed effect of clindamycin and macrolide antibiotics against Toxoplasma gondii tachyzoites (E. R. Pfefferkorn and S. E. Borotz, Antimicrob. Agents Chemother. 38:31-37, 1994), we have carefully examined the replication of parasites as a function of time after drug addition. Intracellular tachyzoites treated with up to 20 M clindamycin (>1,000 times the 50% inhibitory concentration) exhibit doubling times indistinguishable from those of controls (ϳ7 h). Drug-treated parasites emerge from infected cells and establish parasitophorous vacuoles inside new host cells as efficiently as untreated controls, but replication within the second vacuole is dramatically slowed. Growth inhibition in the second vacuole does not require continued presence of drug, but it is dependent solely on the concentration and duration of drug treatment in the first (previous) vacuole. The susceptibility of intracellular parasites to nanomolar concentrations of clindamycin contrasts with that of extracellular tachyzoites, which are completely resistant to treatment, even through several cycles of subsequent intracellular replication. This peculiar phenotype, in which drug effects are observed only in the second infectious cycle, also characterizes azithromycin and chloramphenicol treatment, but not treatment with cycloheximide, tetracycline, or anisomycin. These findings provide new insights into the mode of clindamycin and macrolide action against T. gondii, although the relevant target for their action remains unknown.The protozoan parasite Toxoplasma gondii is a ubiquitous human pathogen long recognized as a source of congenital neurological abnormalities (19). In recent years, this parasite has also acquired considerable notoriety as an opportunistic infection associated with AIDS (16). The ability of T. gondii parasites to persist as latent cysts in the tissues of infected patients mandates chronic treatment for infected AIDS patients, to guard against recrudescence. Unfortunately, the traditional therapeutic regimen of pyrimethamine plus sulfonamides (18) is not always suitable for prolonged treatment, because of the emergence of sulfa hypersensitivity and other adverse side effects (12,14,27).Clindamycin (a lincosamide) and several macrolide antibiotics have proven effective for the treatment of AIDS-toxoplasmosis, usually in combination with pyrimethamine (6, 13, 15). These compounds are known to block protein synthesis in bacteria by interacting with the peptidyl transferase domain of 23S rRNA (5), but their target in T. gondii and related parasites remains unclear (1). Early difficulties in establishing a functional in vitro system to study clindamycin and macrolide action against T. gondii (4, 9, 17) can now be explained by the long lag period between drug administration and effect (20,21). Nanomolar drug concentrations block parasite replication, but only 2 to 3 days after treatment-a remarkable delay, considering that the tachyzoite undergoes ϳ8 generations in this time.In order to more precisely e...

Toxoplasma gondii sporozoites form two parasitophorous vacuoles during development within host cells, the first (PV1) during host cell invasion and the second (PV2) 18 to 24 h postinoculation. PV1 is structurally distinctive due to its large size, yet it lacks a tubulovesicular network (C. A. Speer, M. Tilley, M. Temple, J. A. Blixt, J. P. Dubey, and M. W. White, Mol. Biochem. Parasitol. 75:75-86, 1995). Confirming the finding that sporozoites have a different electron-dense-granule composition, we have now found that sporozoites within oocysts lack the mRNAs encoding the 5 nucleoside triphosphate hydrolases (NTPase). NTPase first appears 12 h postinfection. Other tachyzoite dense-granule proteins, GRA1, GRA2, GRA4, GRA5, and GRA6, were detected in oocyst extracts, and antibodies against these proteins stained granules in the sporozoite cytoplasm. In contrast to tachyzoite invasion of host cells, however, sporozoites did not exocytose the dense-granule proteins GRA1, GRA2, or GRA4 during PV1 formation. Even after NTPase induction, these proteins were retained within cytoplasmic granules rather than being secreted into PV1. Only GRA5 was secreted by the sporozoite during host cell invasion, becoming associated with the membrane surrounding PV1. Microinjection of sporozoite-infected cells with fluorescent dyes showed that PV1 is impermeable to fluorescent dyes with molecular masses as small as 330 Da, indicating that PV1 lacks channels through which molecules can pass from the host cytoplasm into the vacuole. By contrast, lucifer yellow rapidly diffused into PV2, demonstrating the presence of molecular channels. These studies indicate that PV1 and PV2 are morphologically, immunologically, and functionally distinct, and that PV2 appears to be identical to the tachyzoite vacuole. The inaccessibility of PV1 to host cell nutrients may explain why parasite replication does not occur in this vacuole.

Memory retention and transfer in organisms happen at either the neural or genetic level. In humans, addictive behavior is known to pass from parents to offspring. In flatworm planaria (Dugesia tigrina), memory transfer has been claimed to be horizontal, i.e., through cannibalism. Our study is a preliminary step to understand the mechanisms underlying the transfer of addictive behavior to offspring. Since the neural and neurochemical responses of planaria share similarities with humans, it is possible to induce addictions and get predictable behavioral responses. Addiction can be induced in planaria, and decapitation will reveal if the addictive memories are solely stored in the brain. The primary objective was to test the hypothesis that addictive memory is also retained in the brainless posterior region of planaria. The surface preference of the planaria was first determined between smooth and rough surfaces. Through Pavlovian conditioning, the preferred surface was paired with water and the unpreferred surface with sucrose. After the planaria were trained and addicted, their surface preference shifted as a conditioned place preference (CPP) was established. When decapitated, the regenerated segment from the anterior part containing the brain retained the addiction, thus maintaining a shift in the surface preference. Importantly, we observed that the posterior part preserved this CPP as well, suggesting that memory retention is not attributed exclusively to the brain but might also occur at the genetic level. As a secondary objective, the effects of neurotransmitter blocking agents in preventing addiction were studied by administering a D1 dopamine antagonist to planaria, which could provide pointers to treat addictions in humans.